1. Field of the Invention
The invention is in the field of molecular biology as related to the control of programmed cell death.
2. Description of the Background Art
Cell death occurs as a normal aspect of animal development as well as in tissue homeostasis and aging (Glucksmann, A., Biol. Rev. Cambridge Philos. Soc. 26:59-86 (1950); Ellis et al., Dev. 112:591-603 (1991)). Naturally occurring cell death acts to regulate cell number, to facilitate morphogenesis, to remove harmful or otherwise abnormal cells and to eliminate cells that have already performed their function. Such regulated cell death is achieved through a cell-endogenous mechanism of suicide, termed programmed cell death or apoptosis (Wyllie, A. H., in Cell Death in Biology and Pathology, Bowen and Lockshin, eds., Chapman and Hall (1981), pp. 9-34). Programmed cell death or apoptosis occurs when a cell activates this internally encoded suicide program as a result of either internal or external signals. The morphological characteristics of apoptosis include plasma membrane blebbing, condensation of nucleoplasm and cytoplasm and degradation of chromosomal DNA at inter-nucleosomal intervals. (Wyllie, A. H., in Cell Death in Biology and Pathology, Bowen and Lockshin, eds., Chapman and Hall (1981), pp. 9-34). In many cases, gene expression appears to be required for programmed cell death, since death can be prevented by inhibitors of RNA or protein synthesis (Cohen et al., J. Immunol. 32:38-42 (1984); Stanisic et al., Invest. Urol. 16:19-22 (1978); Martin et al., J. Cell Biol. 106:829-844 (1988)).
The genetic control of programmed cell death has been well-elucidated by the work on programmed cell death in the nematode C. elegans. Programmed cell death is characteristic and widespread during C. elegans development. Of the 1090 somatic cells formed during the development of the hermaphrodite, 131 undergo programmed cell death. When observed with Nomarski microscopy, the morphological changes of these dying cells follow a characteristic sequence. (Sulston et al., Dev. Biol. 82:110-156 (1977); Sulston et al., Dev. Biol. 100:64-119 (1983)). Fourteen genes have been identified that function in different steps of the genetic pathway of programmed cell death in this nematode (Hedgecock et al., Science 220:1277-1280 (1983); Ellis et al., Cell 44:817-829 (1986); Ellis et al., Dev. 112:591-603 (1991); Ellis et al., Genetics 112:591-603 (1991b); Hengartner et al., Nature 356:494-499 (1992); Ellis et al., Dev. 112:591-603 (1991)). Two of these genes, ced-3 and ced-4, play essential roles in either the initiation or execution of the cell death program. Recessive mutations in these genes prevent almost all of the cell deaths that normally occur during C. elegans development. Additional support for the view that ced-3 and ced-4 cause cell death comes from the genetic analysis of mosaics (Yuan et al., Dev. Biol. 138:3341 (1990)). The ced-4 gene encodes a novel protein that is expressed primarily during embryogenesis, the period during which most programmed cell deaths occur (Yuan et al., Dev. 116:309-320 (1992)).
A gain-of-function mutation in ced-9 prevents the normal programmed cell death, while mutations that inactivate ced-9 are lethal, suggesting that ced-9 may act by suppressing programmed cell death genes in cells that normally do not undergo programmed cell death (Hengartner, M., et al., Nature 356:494-499 (1992)). The ced-9 gene encodes a protein product that shares sequence similarity with the mammalian proto-oncogene and cell death suppressor bcl-2 (Hengartner, M., et al., Cell 76:665-676 (1994)). The lethality of ced-9 loss- of-function mutations can be suppressed by mutations in ced-3 and ced-4, indicating that ced-9 acts by suppressing the activity of ced-3 and ced-4. Genetic mosaic analyses indicate that ced-3 and ced-4 likely act in a cell-autonomous fashion within dying cells, suggesting that they might be cytotoxic proteins and/or control certain cytotoxic proteins in the process of programmed cell death (Yuan, J., et al., Dev. Bio. 138:33-41 (1990)). The 549 amino acid sequence of the ced-4 protein, deduced from cDNA and genomic clones, contain two regions that are similar to the calcium-binding domain known as the EF-hand (Kretsinger, 1987); however, it is still not clear at present whether calcium plays a role in regulating ced-4 or programmed cell death in C. elegans.